Flagellin, the major component of bacterial flagellae, is recognized by two innate immune detectors: TLR5 and Ipaf. TLR5 responds to extracellular flagellin while Ipaf signals in response to cytoplasmic flagellin. In this proposal, we study the molecular determinants that contribute to flagellin recognition by both TLR5 and Ipaf, and the mechanisms by which extracellular and cytoplasmic recognition of flagellin contributes to bacterial clearance. First, we will solve the crystal structure of TLR5 bound to flagellin and define the molecular determinants within the TLR5/flagellin and the TLR5/TLR5 dimerization interfaces. Mutational analysis of these residues will validate the model and will link structure to function. The role of glycosylation in TLR5 function will also be investigated. Second, we will study the role of TLR5 in host defense against urinary tract infections with E. coli, specifically analyzing the mechanisms by which recognition of extracellular flagellin promotes bacterial clearance, inflammation, and antimicrobial activity. We will also use a number of approaches, including adoptive transfer of bone marrow, to determine the cell types within the bladder and kidney that mediate TLR5-induced bacterial clearance. Third, we will investigate the molecular determinants of flagellin that activate the Ipaf system by mutational analysis, and examine whether, like in the case of TLR5, flagellin from certain strains have evolved to evade the Ipaf system. The direct cytosolic receptor for flagellin will be identified, whether it be Ipaf itself or another molecule that activates Ipaf indirectly. Fourth, we will probe the role of Ipaf in detecting cytoplasmic flagellin and clearing systemic bacterial infection using two approaches. A) We will use an engineered strain of S. typhimurium as a tool to specifically deliver flagellin from the vacuole to the cytoplasm via the type III secretion system. B) We will use L. monocytogenes to examine the innate immune responses activated upon detection of a flagellated cytoplasmic pathogen. In both models, the role of Caspase 1-mediated cytokine release (IL-1beta, IL-18, and, indirectly, IFN-gamma) and Caspase 1-mediated cell death will be compared. Together, these studies will define the structural elements involved in flagellin recognition by TLR5 and the Ipaf system. Further, they will elucidate the role of TLR5 and Ipaf in activating innate immunity, and determine the mechanisms by which these sensors promote clearance of bacterial infection.